Espin distribution as revealed by super-resolution microscopy of stereocilia

Abstract

Auditory hair cells are the mechanical sensors of sound waves in the inner ear, and the stereocilia, which are actin-rich protrusions of different heights on the apical surfaces of hair cells, are responsible for the transduction of sound waves into electrical signals. As a crucial actin-binding and bundling protein, espin is able to cross-link actin filaments and is therefore necessary for stereocilia morphogenesis. Using advanced super-resolution stimulated emission depletion microscopy, we imaged espin expression at the sub-diffraction limit along the whole length of the stereocilia in outer hair cells and inner hair cells in order to better understand espin's function in the development of stereocilia.

Keywords: Espin; stereocilia; super-resolution imaging.

Figure 1

Distribution of espin in the stereocilia of HCs. A. Representative confocal images of espin (red) and F-actin (green) in the OHCs and IHCs from P14 mice. Scale bar, 4 μm. B. Confocal and STED images of espin (red) and F-actin (green) in OHCs and IHCs from the boxed region indicated in (A). Scale bars, 2 μm. C. Magnifications of the boxed regions indicated in (B) and intensity profiles from the corresponding lines are shown beneath.

Figure 2

Distribution of espin in different regions of the cochlea. A. Representative confocal images of espin (red) and F-actin (green) in the apical, middle, and basal turns of the cochlea from P14 mice. Scale bar, 5 μm. B. Confocal and STED images of espin (red) and F-actin (green) in IHCs from the apical, middle, and basal turns of the cochlea. Intensity profiles from the corresponding dashed lines are shown right. Scale bar, 1 μm.

Figure 3

Actin filaments bundled by espin in stereocilia. A. Representative confocal images of espin-mNeonGreen (green), F-actin (red) and DAPI (blue) in the Espin-mNeonGreen transfected cells. White stars indicated the non-transfected cells surrounding the transfected cells. Scale bar, 10 μm. B. Representative confocal images of espin-HA (red) and F-actin (green) in the espin-HA transfected cell. The bright-field image to the lower-right shows the individual cells in the captured region. White stars indicate the non-transfected cells surrounding the transfected cell. Scale bar, 10 μm. C. Co-immunoprecipitation of espin and actin. Human HEK293T cells were transfected with the FLAG, Actb-FLAG, or Espin-HA constructs. Immunoprecipitations were carried out with FLAG antibodies followed by western blotting to detect co-expressed proteins using the HA antibody. Representative images from three independent experiments are shown. D. Representative confocal images of espin-HA (red) and F-actin (green) in the espin-HA-transfected cells, with magnification of the white boxed regions to the right. Scale bars are 4 μm and 1 μm, respectively. The dashed lines are numerically labeled, and the corresponding intensity curves along the dashed lines are shown. E. Transmission electron microscopy image of a stereocilium with its magnification. Red arrows indicate the actin-membrane linkages. Scale bars are 200 nm and 25 nm, respectively. F. Representative STED images of a stereocilium with its magnification, co-labeled with espin (red) and F-actin (green). Yellow arrows indicate the espin punctas between the edge of the actin filaments and the membrane. Scale bars are 200 nm and 100 nm, respectively. G. Representative magnified STED images of stereocilia at different development stages after birth, co-labeled with espin (red) and F-actin (green). Scale bar, 100 nm. H. Functional model of espin in the a stereocilium.

Figure 4

Distribution of espin in deaf mice. A. Representative confocal images of espin (red) and F-actin (green) in the organ of Corti from adult Brg1^-/- and Atoh1-Brg1^-/- mice. Scale bar, 10 μm. B. STED images of espin (red) and F-actin (green) in OHCs and IHCs from adult Brg1^-/- and Atoh1-Brg1^-/- mice. Regions indicated by white closed curves show the cuticular plates. Scale bars, 2 μm.

Figure 5

Altered espin distribution in OHCs from Atoh1-Brg1^-/- mice. A. Representative SEM images of the organ of Corti from adult Brg1^-/- and Atoh1-Brg1^-/- mice. Scale bar, 5 μm. B. Representative confocal images of the organ of Corti from adult Atoh1-Brg1^-/- mice. The espin signal from the same optical field is shown in the insert image. Red, espin. Green, F-actin. Numbers I-V indicate impaired OHCs with different morphologies. White triangle indicates the occasional missing IHC in adult Atoh1-Brg1^-/- mice. Scale bar, 5 μm. C. STED images of espin (red) and F-actin (green) in different OHC morphologies in adult Atoh1-Brg1^-/- mice. Scale bars, 1 μm.